Stabilizing agent for pharmaceutical proteins

ABSTRACT

A method for stabilising a human blood protein or human blood plasma protein with a molecular weight of &gt;10 KDa by adding melezitose to a solution comprising the human blood protein or human blood plasma protein with a molecular weight of &gt;10 KDa.

CROSS REFERENCE TO RELATED APPLICATIONS

The application is a Continuation of U.S. application Ser. No.13/642,020, filed Dec. 27, 2012, which is U.S. National Stage of PCTApplication No. PCT/EP2011/056326, filed Apr. 20, 2011, which claims thebenefit of U.S Provisional Patent Application No. 61/325,975, filed Apr.20, 2010, and which claims the benefit of European Patent ApplicationNo. 10160470.0, filed Apr. 20, 2010, all of which are herebyincorporated by referent in their entirety.

The invention pertains to a method for stabilising a human blood proteinor human blood plasma protein with a molecular weight of >10 KDa, acomposition in solid or liquid state of human blood protein or humanblood plasma protein with a molecular weight of >10 KDa and the use ofmelezitose for stabilisation of a human blood protein or human bloodplasma protein.

The stabilisation of therapeutic proteins is a major challenge for theformulation scientists in the pharmaceutical industry today. There aremany kinds of stresses that can cause both reversible and irreversiblechanges to the proteins, such as aggregation, precipitation ordenaturation. These difficulties call for the need of agents thatstabilize these delicate proteins. Formulation development is a criticalstep, requiring careful selection of excipients to provide a high yieldof protein activity during the purification process as well as duringthe pharmaceutical process and as a final product. In particular this istrue for human blood proteins and human blood plasma proteins.

One of the most widely used stabilizers for protein formulations arecarbohydrates, also called saccharides. Carbohydrates are built oflinked basic carbohydrate components called monosaccharides, and can beof different length and can thus have different characteristics.

Sucrose and trehalose, the two most commonly used stabilizers, are bothdisaccharides, hence composed of two monosaccharides.

As compared to two of the most commonly used carbohydrate stabilizerssucrose and trehalose, which are disaccharides, melezitose is atrisaccharide. It is generally indicated [Wang W, Lyophilisation anddevelopment of solid protein pharmaceuticals, Int J Pharm 203, 1-60,2000; Carpenter J. F., Chang B. S., Garzon-Rodriguez W., Randolph T. W.,Rational design of stable lyophilized protein formulations: Theroy andpractice, chapter 5, ed Carpenter and Manning, Kluiwer Academic/PlenumPublishers, New York, 2002] that disaccharides are the first choice forstabilisation of proteins both in solution and in lyophilized state.Some disaccharides, such as lactose or maltose, are reducing sugars thatcan degrade proteins via the Malliard reaction during storage in thesolid state. If larger saccharides are used as stabilizers inlyophilized preparations, literature suggests that these are lessefficient due to steric hinderance of the protein-stabilizer interaction[Carpenter J. F., Chang B. S., Garzon-Rodriguez W., Randolph T. W.,Rational design of stable lyophilized protein formulations: Theroy andpractice, chapter 5, ed Carpenter and Manning, Kluiwer Academic/PlenumPublishers, New York, 2002].

The review article Wang, W., International Journal of Pharmaceutics, 203(2000) 1-60, “Lyophilization and development of solid proteinpharmaceuticals”, discloses i.a. that maltose, glucose and maltotriosecould increase the recovery of catalase activity at 1 mg ml⁻¹, butmaltopentaose, maltohexaose, and maltoheptaose were not as effective.The ineffectiveness of larger saccharides suggests that proteinstabilization by sugars may depend on the glucoside side chain length ofthe sugar that may interfere with intermolecular hydrogen-bondingbetween stabilizing sugars and proteins. This review article recommendsdisaccharides as stabilizers (p. 9/10).

WO-A-2003/086443 discloses the use of carbohydrates including stachyose,melezitose, and various mono- and disaccharides for preparation ofintranasally administerable polypeptide preparations. The sugars serveas agents to reduce the effects of shear stress during spraying.

WO-A-86/04486 discloses chromatographic purification of i. a. factorVIII wherein melezitose is used as a hydration additive during thechromatographic process.

WO-A-91/18091 discloses a method of preserving delicate biologicalsubstances or organic compounds (a) in a dry state and/or (b) atelevated temperatures and/or (c) under irradiation comprisesincorporating in a system containing the said substances or compounds asugar or a sugar derivative selected from (i) a non reducing glycosideof a polyhydroxy compounds selected from sugar alcohols and otherstraight chain polyalcohols, or (ii) a non-reducing oligosaccharideselected from raffinose, stachyose and melezitose.

Mollmann, S. H. et al reports in Drug Dev. Ind. Pharm. 2006 July;(6):765-78 about the stability of insulin in solid formulationscontaining melezitose and starch.

The present invention provides a method for stabilising a human bloodprotein or human blood plasma protein with a molecular weight of >10 KDaby adding melezitose to a solution comprising the human blood protein orhuman blood plasma protein with a molecular weight of >10 KDa.

Preferably, the human blood protein or human blood plasma protein has amolecular weight of >10 KDa. More preferably, the molecular weight is inthe range of 10 KDa to 300 KDa. Most preferably, the molecular weight isin the range of 20 KDa to 200 KDa. It may be advantageous that the humanblood protein or human blood plasma protein has a molecular weight rangeof 50 KDa to 100 KDa or a molecular weight range of 100 KDa to 150 KDaor a molecular weight range of 150 KDa to 200 KDa.

In particular, the human blood protein or human blood plasma proteinwith a molecular weight of >10 KDa is a pharmaceutically or biologicallyrelevant protein. The pharmaceutically or biologically relevant humanblood protein or human blood plasma protein with a molecular weightof >10 KDa which can be stabilised according to the invention can be arecombinantly produced human blood protein or human blood plasmaprotein.

The term “protein” includes chemically synthesised proteins as well asnaturally synthesised proteins which are encoded by genes of cultivatedcell as well as recombinant proteins secreted by cells. Recombinantproteins are those which are encoded by transgenes introduced into thecells by molecular biology techniques. Proteins can be modified bychemical methods or by enzymes in post translatorial processes.

In accordance with the invention, “protein” includes proteins of humanin particular those produced by cell-cultures, but also proteins ofother sources such as plants, insects, etc., and mutated, artificial,synthetic, fusion or chimeric proteins.

The term “human blood protein or human blood plasma protein with amolecular weight of >10 KDa” includes in particular human blood clottingfactors including fibrinogen, fibrin monomer, prothrombin, thrombin, FV,FVa, FX, FXa, FIX, FIXa, FVII, FVIIa, FVIII, FXI, FXIa, FXII, FXIIa,FXIII, FXIIIa, von Willebrand factor, ADAMTS13 etc., transport proteinssuch as albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobin,hemopexin, etc., protease inhibitors such as β-antithrombin,α-antithrombin, α2-macroglobulin, Cl-inhibitor, tissue factor pathwayinhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3),Protein C, Protein S, Protein Z, etc., immunoglobulin's such aspolyclonal antibodies (IgG), monoclonal antobodies, IgG1, IgG2, IgG3,IgG4, IgA, IgA1, IgA2, IgM, IgE, IgD, Bence Jones protein etc., cellrelated plasma proteins such as fibronectin, thromboglobulin, plateletfactor4, etc., apolipoproteins such as apo A-I, apo A-II, apo E,complement factors such as Factor B, Factor D, Factor H, Factor I,C3b-Inactivator, properdin, C4-binding protein etc., growth factors likePlatelet derived growth factor (PDGF), Epidermal growth factor (EGF),Transforming growth factor alfa (TGF-α), Transforming growth factor beta(TGF-β), Fibroblast growth factor (FGF) and Hepatocyte growth factor,antiangionetic proteins such as latent-antithrombin andprelatent-antithrombin etc., highly glycosylated proteins includingalfa-1-acid glycoprotein, antichymotrypsin, inter-α-trypsin inhibitor,α-2-HS glycoprotein, C-reactive protein, and other human blood proteinsor human blood plasma proteins with a molecular weight of >10 KDa suchas histidine-rich glycoprotein, mannan binding lectin, C4-bindingprotein, fibronectin, GC-globulin, plasminogen, α-1 microglobulin,C-reactive protein, blood factors such as erythropoietin, interferon,tumor factors, tPA, gCSF and derivatives and muteins thereof.Particularly of interest is factor IX, factor VIII, G-CSF, vWF,antithrombin (AT), Hepatocyte Growth Factor (HGF), polyclonal IgG,alfa-1 antitrypsin, Factor H, Factor I, C1-esterase inhibitor, FactorVII and combinations thereof. However, polypeptides such as insulin arenot covered by the term “Human blood protein or human blood plasmaprotein with a molecular weight of >10 KDa” simply because insulin has amolecular weight of about 5.700 Da.

The terms “human blood protein” and “human blood plasma protein” includederivatives, especially molecules that have been modified to have anextended half-life. Modifications for half-life prolongation include,but are not limited to, fusion proteins, proteins modified bymutagenesis and proteins linked to a conjugate by covalent ornon-covalent binding. According to the invention the human blood plasmaproteins or human blood proteins may be covalently coupled to hydroxylethyl starch (HES) molecules, in particular providing molecules with amolecular weight of 20 to 200 KDa.

Where reference is made to the molecular weight, this refers to themolecular weight of the compounds (i.e. including the molecular weightof any chemical compound covalently coupled to the protein).

The present invention provides in particular a method wherein thesolution is transferred into the solid state.

This invention relates to the finding of a new stabilizing agent for apharmaceutical human blood protein or human blood plasma protein with amolecular weight of >10 KDa.

Surprisingly, it has been found that melezitose can be used asstabilizer for human blood proteins or human blood plasma proteins witha molecular weight of >10 KDa, such as recombinant factor VIII (170KDa), factor IX (55 kDa) and HESylated G-CSF (120 kDa). It is expectedthat human blood proteins and human blood plasma proteins of similarmolecular weight will have similar stabilization requirements. Forexample, factor IX is a vitamin k dependent human blood plasma proteinand has biochemical similarities with all other vitamin K dependenthuman blood plasma proteins. The Gla domain is a common structuralfeature in all these vitamin K-dependent proteins and immediately afterthe Gla domain, each of the proteins (except prothrombin) has one ormore EGF-like domains. The vitamin K-dependent proteins require Ca²⁺ions to express their physiological function and the calcium bindingsites involve at least the Gla domain and the EGF-like domains. Calciumbinding enables these proteins to bind to phospholipids/cell membranesand thus express their full biological activities. Seven human bloodplasma proteins are known to be dependent on vitamin K for theirbiosynthesis. They are prothrombin (factor II, 72 KDa), factorVII/factor VIIa (50/50 KDa), factor IX (55 KDa) or factor IXa, factor X(59 KDa) or factor Xa, protein C (62 KDa), protein S (69 KDa) andprotein Z (62 KDa).

Surprisingly it has been found that human blood proteins and human bloodplasma proteins which are covalently bound with a hydroxylethyl starch(HES) molecule with a molecular weight in the range of 20-200 KDa aresuitable for stabilization with Melezitose.

Melezitose has shown an excellent ability to maintain the proteinactivity in both lyophilized formulations and in solution.

According to the invention the step of transferring the solution intothe solid state is lyophilisation upon adding of melezitose. Melezitosemay also be used in combination with other sugars, such as trehalose, orsucrose. Melezitose, also spelled melicitose, is a nonreducingtrisaccharide sugar that is produced by many plant sap eating insects,including aphids such as Cinara pilicornis by an enzyme reaction. TheIUPAC name is(2R,3R,4S,5S,6R)-2-[[(2S,3S,4R,5R)-4-hydroxy-2,5-bis(hydroxymethyl)-3-[[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxylmethyl)-2-tetrahydropyranyl]oxy]-2-tetrahydrofuranyl]oxy]-6-(hydroxylmethyl)-tetrahydropyran-3,4,5-triol.

Melezitose has a molecular weight of 504.44 g/mol.

The respective structure is represented by the formula:

Typically melezitose is present in an amount of up to about 1000 mM. Thelower limit depends on the amount of melezitose resulting in asufficient stabilizing effect on the protein of interest. The suitableamount can be readily determined by the skilled person employing themethodology of the examples and his or her general knowledge. A feasiblerange is for example 10 mM to about 200 mM or from about 10 mM to about100 mM related to the final formulation.

Preferably the amount larger than 20 mM or larger than 30 mM.

Preferably the amount of melezitose per amount of human blood protein orhuman blood plasma protein is in the range of 10:1 to 5000:1, preferablyin the range of 50:1 to 150:1, or 1000:1 to 3000:1 calculated on aweight per weight basis, i.e. the amount of melezitose is higher thanthe amount of the protein.

In a preferred embodiment, 10 to 100 mg Melizitose are included in onepharmaceutical dosage of a protein.

Subject of the present invention is a composition comprising a humanblood protein or human blood plasma protein with a molecular weightof >10 KDa and melezitose. The composition may be present in the liquidor solid state.

In an embodiment of the invention the composition of the invention iscomprising further a bulking agent, a surface active agent, a bufferingagent, a further stabilizer and/or tonicity modifier.

A surfactant according to the invention is a compound that adsorbs tosurfaces and interfaces and thereby counteracts activity loss of aprotein due to adsorption. This type of activity loss may occur duringthe entire pharmaceutical processing as well as while handling thereconstituted product prior to and during administration to a patient.Commonly used surfactants are Polysorbate 80, Polysorbate 20 andpoloxamers, in particular Poloxamer 188. Also proteins such as albumin,in particular recombinant albumin can be used as a surface active agent.Also recombinant albumin may be used according to an embodiment of theinvention.

A pH buffering agent is referred to as a compound with a bufferingcapacity in the optimal pH range of the protein to be formulated. Thepresent invention, when appropriate, embodies sodium citrate, maleicacid, histidine, 2-(4-(2-hydroxy-ethyl)-1-piperazinyl)-ethan sulfonicacid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS),2-(N-morpholino)ethanesulfonic acid (MES),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), orTris(tris(hydroxymethyl)aminomethane) as a pH buffering agent. Thebuffering agent is present in an amount to maintain a pH in a range inwhich the protein stays functional. This is different from one proteinto another. The skilled person knows about the preferred ranges of therespective protein, in particular of human blood proteins or human bloodplasma proteins with a molecular weight of >10 KDa. As an example,sodium citrate keeps the pH ranging from 6.5 to 7.5. A suitable form ofthe sodium citrate is the dihydrate form. Generally, the compositionsaccording to the invention can be in lyophilized form, but are alsorepresented by solutions such as a solution to be lyophilized and asolution reconstituted from a lyophilized composition.

A tonicity modifier is referred to as a compound that is present in theformulation to balance tonicity. The present invention, whereappropriate, embodies sodium chloride, arginine, glycine, potassiumchloride, sugars or sugar alcohols as tonicity modifiers.

Although melezitose exhibits cryo- and lyoprotecting properties,additional cryo- and lyoprotectant (cryo-/lyoprotectant), may also bepresent. This is a compound present in the formulation to furtherdecrease or prevent loss of protein activity during the freezing anddrying steps of a lyophilization process and during subsequent storageof the lyophilized product. The present invention, where appropriate,embodies non-reducing disaccharides such as sucrose and trehalose, andreducing disaccharides, such as maltose and lactose, as additionalcryo-/lyoprotectants.

A bulking agent is referred to as an excipient present in theformulation to provide mechanical support to the lyophilized cake and toincrease the dry weight. The bulking agent can either be in acrystalline state, as sodium chloride, or in an amorphous state, asarginine. The amount of the bulking agent can be up to 10% by weightbased on the final formulation. The present invention, whereappropriate, embodies sodium chloride, glycine, mannitol, sucrose orarginine as bulking agent.

A further subject of the present invention is the use of melezitose forlong term stabilisation of a protein in the dried state such aslyophilised formulations for at least 6 months, in particular at least12 months, more particular at least 18 months, still more particular 24months.

The invention is further described in the following non-limitingexamples.

Activity Analysis—Factor VIII

The factor VIII activity was measured with a chromogenic assay or withthe one stage assay and the unit of factor VIII was expressed inInternational Units (IU).

The chromogenic assay is the method prescribed in the EuropeanPharmacopoeia. The method is a two-stage photometric method thatmeasures the biological activity of factor VIII as a cofactor. FactorVIII activates factor X into factor Xa, which in turn is enzymaticallycleaved into a product that can be quantified spectrophotometrically.

The one-stage clotting assay is based on the ability of a factor VIIIcontaining sample to correct the coagulation time of factor VIIIdeficient plasma in the presence of phospholipid, contact activator andcalcium ions. The time of appearance of a fibrin clot is measured in onestep.

Activity Analysis—Factor IX

The biological activity of factor IX was measured with a one-stageclotting assay and/or a chromogenic assay and the unit of factor IX wasexpressed in International Units (IU) as defined by the current WHOfactor IX concentrate standard.

The one-stage clotting assay is the method prescribed in the EuropeanPharmacopoeia. The principle of the assay is based on the ability of afactor IX containing sample to correct the coagulation time of a factorIX deficient plasma in the presence of phospholipids, contact activatorand calcium ions. The time of appearance of a fibrin clot is measured inone step. The factor IX activity is inversely proportional to thecoagulation time.

The chromogenic assay is a two-stage photometric method. In the firststage, factor IX is activated to factor IXa by activated factor XI (XIa)in the presence of thrombin, phospholipids an calcium. Factor IXa formsan enzymatic complex with thrombin activated factor VIII (VIIIa) that inthe presence of phospholipids and calcium activates factor X into factorXa. In stage two, factor Xa hydrolyses a factor Xa specific chromogenicsubstrate thus liberating a chromophoric group pNA that can bequantified spectrophotometrically. The factor IX activity is directlyproportional to the amount of generated factor Xa.

Analysis—Recombinant HESylated G-CSF

Resource S HPLC Analysis of HES-G-CSF

The samples are diluted to 0.1 mg/mL with eluent A. 20 μg are injectedonto a Resource S 1 mL column (GE Healthcare, Munich, Germany).

Eluent A: 20 mM Na Acetate, pH 4.0

Eluent B: 20 mM Na Acetate, 0.5 M NaCl, pH 4.0

Flow rate: 1 mL/min

Gradient: 0%-8%   1.8-2.0 min 8%-52%  2.0-13.0 min 52%-100% 13.0-13.6min

The peak width of the HES-G-CSF peak is taken as quality criterion as itwas shown that aggregated HES-G-CSF has a bigger peak width. The gain ofpeak width is defined as the difference of the HES-G-CSF peak widthbefore and after thermal or shear stress

EXAMPLES

Recombinant Factor VIII

The factor VIII used in the experiments is a recombinant human B-domaindeleted factor VIII protein, produced in the human cell line HEK293Faccording to the process described in EP-A-1 739 179 (Schröder et al).The purification process consisted of five chromatography steps andgenerated a highly pure factor VIII protein preparation (Winge et al,WO-A-2009/156430) with a human glycosylation like pattern (Sandberg etal, PCT/EP2009/060829).

Plasma Derived Factor IX

The material used in these experiments origins from the commerciallyavailable product Nanotiv®, which is a high purity SD treated andnanofiltered factor IX concentrate. Before use in these experiments thematerial has been further purified over a gel filtration column wherethe factor IX monomer peak was used for further experiments.

Recombinant HESylated G-CSF

The cell line used is a derivative of human embryonic kidney cell 293(HEK 293), which was adapted to serum-free growth. This host, HEK 293F,was stably transfected with an expression cassette carrying the cDNAcoding sequence for G-CSF. The strong promoter was used for thecassette. The general process is also described in EP 1739179 (Schröderet al).

The purification process consisted of four chromatography steps andgenerated a highly pure G-CSF protein preparation. The G-CSF protein wascoupled to a hydroxylethyl starch (HES) derivative of a molecular weightof approximately 100 KDa. Finally, the HES-G-CSF was purified from thenon reacted HES derivative and G-CSF by one chromatography step,resulting in a molecule with a total molecular weight of approximately120 KDa.

Example 1 Stabilisation of rFVIII by Melezitose in Solution

Preparation

The recombinant factor VIII (rFVIII) was prepared according to thedescription in the experimental section above. This experiment comparedthe stabilizing effect of melezitose on rFVIII in solution, with that ofthe commonly used stabilizer trehalose. The concentration of rFVIII was100 IU/ml. The compositions of the formulations investigated in thisexperiment are displayed in Table 1.

TABLE 1 Compositions of the formulation. 1A 1B Melezitose, mM — 48Trehalose dihydrate, mM 63 — NaCl, mg/ml 30 30 Calcium chloridedihydrate, 0.5 0.5 mg/ml Poloxamer 188, mg/ml 2 2 Histidine, mg/ml 3 3

The formulations were stored for up to 7 days at +25° C. to evaluate theprotein activity over time. Samples were taken at regular intervals andanalyzed with the chromogenic assay, as described in the experimentalsection above. The results are summarized in Table 2, as percentage ofthe initial value.

TABLE 2 Results. Factor VIII activity over time (days), (% of initialvalue) 0 1 7 1A +25° C. 100 86 85 1B +25° C. 100 82 86

Conclusions of Example 1

This experiment shows that, surprisingly, melezitose, despite of itslower molar concentration, has a stabilizing effect on rFVIII insolution equal to that of trehalose.

Example 2 Stabilisation of rFVIII by Melezitose in Lyophilized Form

Preparation

The recombinant factor VIII (rFVIII) was prepared according to thedescription in the experimental section above. This experiment comparedthe stabilizing effect of melezitose with that of the commonly usedstabilizer trehalose, over the freeze-drying process and in lyophilizedformulations. The concentration of rFVIII was 100 IU/ml. Thecompositions of the formulations investigated in this experiment aredisplayed in Table 3.

TABLE 3 Compositions of the formulations. 2A 2B Trehalose, mM 63 —Melezitose, mM — 48 NaCl, mg/ml 30 30 Calcium chloride dihydrate, 0.50.5 mg/ml Poloxamer 188, mg/ml 2 2 Histidine, mg/ml 3 3

1.5 ml aliquots of the solutions were lyophilized in a laboratory scalefreeze-drier. The protein recovery over the lyophilisation step was 93%for formulation 2B and 86% for formulation 2A. The lyophilized sampleswere stored for up to 4 weeks at +25° C. and +40° C. to evaluate theprotein activity over time. The samples were reconstituted in 1.5 mlwater for injections and analyzed with the chromogenic assay, describedin the experimental section above. Results are summarized in Table 4.

TABLE 4 Results Factor VIII activity over time (weeks), (% of initialvalue) 0 1 2 4 2A +25° C. 100 97 89 * +40° C. 100 98 90 93 2B +25° C.100 117 95 * +40° C. 100 104 97 99 * no significant change

Conclusions of Example 2

Surprisingly, this experiment shows that melezitose is able to protectrFVIII in lower molar concentration than trehalose over thelyophilisation step, and that it stabilizes rFVIII better than trehaloseduring storage.

Example 3 Stabilisation of rFVIII by Melezitose in Lyophilized Form

Preparation

The recombinant factor VIII (rFVIII) was prepared according to thedescription in the experimental section above. This experiment comparedthe stabilizing effect of melezitose at different concentrations overthe freeze-drying process and in lyophilized formulations, and alsocompared the stabilizing effect with the tetrasaccharide Stachyose. Theconcentration of rFVIII was 170 IU/ml. The compositions of theformulations investigated in this experiment are displayed in Table 5.

TABLE 5 Compositions of the formulations. 3A 3B 3C 3D Melezitose, mM 4836 24 — Stachyose, mM — — — 30 NaCl, mg/ml 30 30 30 30 Calcium chloridedihydrate, 0.5 0.5 0.5 0.5 mg/ml Poloxamer 188, mg/ml 2 2 2 2 Sodiumcitrate, mg/ml 2 2 2 2

1.5 ml aliquots of the solutions were lyophilized in a laboratory scalefreeze-drier. The protein recovery over the lyophilisation step was 91to 100% for formulations containing melezitose, while the recovery was84% for formulation 3D containing stachyose as stabilizer. Thelyophilized samples were stored for up to 12 months at +25° C. and +40°C. to evaluate the protein activity over time.

The samples were reconstituted in 1.5 ml water for injections andanalyzed with the chromogenic assay, described in the experimentalsection above. Results are summarized in Table 6.

TABLE 6 Results. Factor VIII activity over time (months), (% of initialvalue) 0 1 2 3 6 12 3A 25° C. 100 * n.a. * * 91 40° C. 100 96 93 93 n.a.n.a. 3B 25° C. 100 92 n.a. 96 95 78 40° C. 100 90 79 73 n.a. n.a. 3C 25°C. 100 91 n.a. 86 86 67 40° C. 100 70 58 48 n.a. n.a. 3D 25° C. 100 95n.a. 79 65 n.a. 40° C. 100 74 n.a. 51 n.a. n.a. n.a. = not analysed; *no significant change

Conclusions of Example 3

This experiment shows that melezitose functions exceptionally well as astabilizer for rFVIII over the lyphilization step and in lyophilizedform. Also, it shows that stachyose is not a preferable stabilizer forlyophilized formulations, as it shows very unsatisfactory results duringstorage at both 25° C. and at 40° C., compared to the melezitosecontaining formulations.

Example 4 Stabilisation of Plasma Factor IX by Melezitose in LyophilizedForm

Preparation

The plasma derived factor IX (pFIX) was prepared according to thedescription in the experimental section above. This experimentinvestigates the stabilizing effect of melezitose on pFIX. Theconcentration of pFIX was 100 IU/ml. The composition of the formulationinvestigated in this experiment is displayed in Table 7.

TABLE 7 Composition of the formulation. 4A Melezitose, mM 42 NaCl, mg/ml30 Polysorbate 80, mg/ml 0.1 Sodium citrate, mg/ml 2.35

1.5 ml aliquots of the solutions were lyophilized in a laboratory scalefreeze-drier. The lyophilized samples were stored for up to 6 months at+5° C., +25° C. and +40° C. to evaluate the protein activity over time.The samples were reconstituted in 1.5 ml water for injections andanalyzed with the chromogenic assay, described in the experimentalsection above.

Results

The protein recovery over the lyophilisation step was about 100%. Theresults of the stability study are shown in Table 8.

TABLE 8 Results. Factor IX activity over time (months), (% of initialvalue) 0 1 3 6 4A  5° C. 100 88 87 85 25° C. 100 94 89 86 40° C. 100 9392 n.a. n.a. = not analysed; * no significant change

Conclusions of Example 4

This experiment shows that, surprisingly, melezitose functions well as astabilizer for factor IX in lyophilized form.

Example 5 Stabilization of HESylated Recombinant G-CSF by Melezitose inLyophilized Form

Preparation

The recombinant HESylated G-CSF (rHES-G-CSF) was prepared according tothe description in the experimental section above. The experimentcompared the stabilizing effect of melezitose on rHES-G-CSF inlyophilized form, with that of the commonly used stabilizer trehalose.The concentration of rHES-G-CSF was 0.3 mg/ml and the compositions ofthe formulations investigated are displayed in Table 9.

TABLE 9 Compositions of the formulation. 5A 5B Melezitose, mM 70 —Trehalose, mM — 70 NaCl, mg/ml 30 30 Polysorbate 20, mg/ml 0.2 0.2Histidine, mg/ml 3 3

1.5 ml aliquots of the solutions were lyophilized in a laboratory scalefreeze-drier. The protein recovery was measured after 4 weeks storage at+40° C. by the Resource S method, described in the experimental sectionabove. Results are summarized in Table 10.

TABLE 10 Results. Gain of peak width (min) after 4 weeks 5A +40° C. 0.045B +40° C. 0.06

Conclusions of Example 5

This experiment shows that melezitose has a stabilizing effect onrHES-G-CSF better than that of the commonly used stabilizer trehalose atequal molar concentration.

Example 6 Stabilisation of Plasma Factor IX by Melezitose Over theFreeze-Drying Step

Preparation

The plasma derived factor IX (pFIX) was prepared according to thedescription in the experimental section above. This experimentinvestigates the stabilizing effect of melezitose on pFIX over thefreeze-drying step, compared to the tetrasaccharide stachyose. Theconcentration of pFIX was 100 IU/ml. The compositions of theformulations investigated in this experiment are displayed in Table 11.

TABLE 11 Compositions of the formulations. 6A 6B Melezitose, mM 42 —Stachyose, mM — 30 NaCl, mg/ml 30 30 Polysorbate 80, mg/ml 0.1 0.1Sodium citrate, mg/ml 2.35 2.35

1.5 ml aliquots of the solutions were lyophilized in a laboratory scalefreeze-drier. The samples were analyzed with the chromogenic assay,described in the experimental section above, before and after thelyophilization step.

Results

The protein recovery over the lyophilisation step was about 100% forformulation 6A, while the corresponding recovery for formulation 6B was84%.

Conclusions of Example 6

This experiment shows that, melezitose functions well as a stabilizerfor factor IX over the lyophilization step. However, stachyose is not asuitable candidate as stabilizer since a significant activity lossoccurs over the freeze-drying step.

The invention claimed is:
 1. A method for the preparation of a human factor VIII-containing product, wherein the method comprises the following steps: (a) providing a human factor VIII-containing solution; (b) adding between 10 mM and 200 mM of melezitose to the human factor VIII-containing solution of step (a), wherein the amount of melezitose per amount of human factor VIII is in the range of 735:1 to 2500:1 when calculated on a weight by weight basis, (c) lyophilizing the solution of step (b) to prepare a lyophilized human factor VIII-containing product, and (d) resuspending the lyophilized human factor VIII-containing product of step (c) to prepare a resuspended human factor VIII-containing solution; wherein the human factor VIII is a human B-domain deleted factor VIII protein recombinantly produced in cell culture, and wherein the human factor VIII is stabilized by the addition of the melezitose in the human factor VIII-containing solution of step (b), in the lyophilized human factor VIII-containing product of step (c), and/or in the resuspended human factor VIII-containing solution of step (d).
 2. The method of claim 1 wherein at least one surface active agent selected from the group consisting of recombinant albumin, Polysorbate 80, Polysorbate 20 and a Poloxamer is present.
 3. The method of claim 1 wherein at least one buffering agent selected from the group consisting of histidine, sodium citrate, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ehtanesuflonic acid (HEPES), Tris, 3-morpholinorpoane-1-sulfonic acid (MOPS) and 1,4Piperazinediethanesulfonic acid (PIPES) is present.
 4. The method of claim 1 wherein at least one further stabilizer selected from the group consisting of sugars, amino acids, polyols, and combinations thereof is present; and/or wherein at least one tonicity modifier selected from the group consisting of sodium chloride, arginine, glycine, potassium chloride, sugars and sugar alcohols is present; and/or wherein at least one bulking agent selected from the group consisting of glycine, mannitol, sodium chloride, arginine and sucrose is present.
 5. The method of claim 1 wherein the lyophilized human factor VIII-containing product of step (c) has improved long-term stability and a shelf-life of at least 6 months.
 6. The method of claim 1, wherein the melezitose is present in an amount from about 10 mM to about 100 mM.
 7. The method of claim 2, wherein the Poloxamer is Poloxamer
 188. 8. The method of claim 5, wherein the lyophilized human factor VIII-containing product of step (c) has improved long-term stability and a shelf-life of at least 12 months.
 9. The method of claim 5, wherein the lyophilized human factor VIII-containing product of step (c) has improved long-term stability and a shelf-life of at least 24 months.
 10. The method of claim 5, wherein the lyophilized human factor VIII-containing product of step (c) has improved long-term stability and a shelf-life of at least 36 months.
 11. The method of claim 1 wherein the human factor VIII protein is not a chimeric human factor VIII protein, a human factor VIII fusion protein or a human factor VIII derivative coupled with one or more chemical compounds. 